SUMMARY HIV-1 envelope (Env) probes are key to the isolation of broadly neutralizing antibodies (bnAbs) and the HIV-1 bnAbs are particularly useful to inform and guide Env immunogen design. As current probes and methods have limitations that hinder efficient bnAb isolation from large numbers of individuals, we propose to develop novel VSV-based probes to display dense HIV-1 Env trimers on the surface to isolate bnAbs. We hypothesize that properly constructed gp140.VSV probes provide a powerful tool for bnAb isolation and allow efficient identification of bnAbs from a large number (n>50) of clade-B and non-B clade infected individuals, some with undefined novel epitopes. To test this hypothesis, we collaborate with Dr. Andres Finzi to study samples from a clade-B infected cohort based in Montreal, Canada, and with Dr. Phillipe Nyambi to study samples from a multi-clade infected cohort based in Yaounde, Cameroon. We have screened 111 Montreal and 260 Cameroon plasmas and identified 32 and 13 donors with bnAb activity, respectively. We propose to 1) construct, characterize, and optimize gp140.VSV probes for various clade-B and non-B Envs, 2) apply gp140.VSV probes to isolate bnAbs from >50 clade-B and non-B clade infected broad neutralizers, and 3) determine the epitopes of newly isolated bnAbs and define new bnAb genetic compositions. In pilot experiments, we generated a gp140.VSV probe displaying the HIV-1 AD17 Env trimer on the surface; using this probe, we recovered two distinct and novel bnAbs from a clade-B infected Montreal donor. This preliminary result supports our hypothesis and provides a proof-of-concept that the gp140.VSV probe can lead to the isolation of novel bnAbs. We anticipate that >50 new bnAbs will be identified by this probing method and some of them will define new Env targets and bnAb genetic compositions. The addition of these bnAbs will complement others to expand the bnAb repertoire and advance our understanding of the bnAb epitopes and genetic compositions, thus facilitating Env immunogen design and post-immunization analysis.